Primary transcripts in higher eukaryotes often contain intervening sequences which must be precisely excise to generate functional messenger RNAs. Nuclear pre-mRNA splicing is thus an essential step in regulating gene expression in every eukaryotic cell. Regulated and alternative splicing play a role in determining normal cell development and generate a broad spectrum of genetic diversity in higher eukaryotes. Aberrant splicing is associated with certain diseases;f or examples, disruption of splicing patterns has been implicated in the oncogenic activation of c-Hras, one of the most commonly mutated genes inhuman cancer. Although much progress has been made in defining the general features of splicing as well as identifying specific components, understanding the regulation of this process will require the analysis of the splicing machinery of the molecular level. Elucidation of RNA-RNA and RNA-protein interactions and the way in which conformational changes are achieved in the spliceosome is central to this understanding. Insights into these questions can be gained by studying the molecular interactions of ATPases, such as PRP16, known to function at specific steps of splicing. This proposal presents experiments to delineate the molecular mechanism by which the protein factor PRP16 uses ATP hydrolysis to promote the final steps of the splicing reaction leading to the formation of mature RNA. The functional domains of the PRP16 protein will be determined by mutational analysis. These studies are important to determine the PRP16 cycle in the splicing reaction by freezing it at distinct pints (e.g. ATP-binding, hydrolysis, binding to or release from the spliceosome). Mutants that prove to be defective in certain functions will provide valuable tools for subsequent genetic and biochemical studies, aimed at isolating novel splicing factors. The choice of yeast as a system permits the powerful combination of generic and biochemical approaches in studying the molecular interactions in splicing. Because the splicing process is evolutionary conserved, insights gained in yeast will have important implications for gene expression in higher eukaryotes. Studies concerning the mechanism of PRP16 function are of particular interest because this protein appears to be a paradigm for class of related factors, members of the DEAH box family. These are related to the superfamily of DEAD (asp-glu-ala-asp) box proteins, some members of which are demonstrated RNA-dependent ATPases and RNA helicases. It is a fascinating possibility that each of the spliceosomal DEAH proteins, like PRP16, uses ATP hydrolysis to promote a conformational change in the spliceosome at a distinct step in splicing. Thus, investigations of PRP16 should offer valuable hints for understanding the role of the other members of the family.